Inhalation of the intracellular gram-negative bacterium Francisella tularensis is highly fatal in humans. Since F. tularensis can be easily weaponized into an aerosolized form, it is considered to be one of the most important potential biowarfare agents. The virulence determinants and protective antigens of F. tularensis have been poorly characterized. To date, the F. tularensis live vaccine strain (LVS), which is derived from F. tularensis subspecies holarctica (type B), has demonstrated varying degrees of protection in humans. However, the protective antigens expressed by the LVS strain have yet to be characterized, and attenuated LVS is unlikely to pass approval for current safety standards for vaccines. Therefore, development of a licensed subunit vaccine, which is currently not available, is of utmost importance. It is our long-term goal to identify candidate T cell epitopes of F. tularensis in "humanized" HLA-DR transgenic mice that can be used to develop subunit vaccines to induce protective immunity in humans. The specific hypothesis underlying this application is that protective T cell epitopes of F. tularensis can be identified in "humanized" HLA-DR transgenic mice that are also processed and presented by human APCs and could be used for the design of a subunit vaccine. We base this hypothesis on the following observations: First, protection to Francisella infection appears to be critically dependent on specific T cell immunity and T cell-derived production of IFNg. Second, vigorous T cell proliferation and cytokine production was detected in HLA-DR4 tg mice upon vaccination with LVS. Third, T cell epitopes of self- and microbial antigens identified in the HLA-DR4 transgenic mice are relevant to humans. We base this assumption on our preliminary data showing that T cell epitopes characterized in "humanized" HLA-DR4 transgenic mice are processed and presented by human HLA-DR4+ APCs. Thus, overall, we believe that the HLA transgenic animals provide a novel tool and model to identify T cell determinants of F. tularensis that have potential use for the development of vaccines for humans. We will test this hypothesis with the following specific aims: Specifically, we will (1) characterize the F. tularensis proteins that induce protective T cell immunity in HLA-DR4 (DRB1*0401) ransgenic mice. (2) Identify protective T cell peptide epitopes in the HLA-DR tg mice. (3) Examine whether epitopes presented by the HLA-DR tg mice are also presented by human APCs.